Recent efforts in this laboratory have developed polyacrylamide gel separation procedures for the study of the molecular events involved in the biosynthesis and export of secretory proteins from the exocrine pancreas. Using the two-dimensional gel technique (isoelectric focusing followed by SDS gradient gel electrophoresis) we have studied the secretory pathway in the guinea pig pancreas on a protein specific basis and discovered (a) asynchrony in the intracellular transport of secretory proteins and (b) postsegregational processing of three prosecretory proteins: protrypsinogen, proamylase, and proelastase. We will now define the molecular characteristics and localize the intracellular sites responsible for asynchronous intracellular transport and postsegregational processing of prosecretory proteins. Using one-dimensional polyacrylamide gel electrophoresis in SDS we will conduct further studies into the molecular events responsible for synthesis of pre-secretory and secretory proteins in the dog pancreas. Studies to date have defined these relationships, which include proteolytic processing and segregation of the nascent polypeptide chain, for only a single secretory protein, trypsinogen 2. Recently improved in-vitro translation techniques and the use of specific immunoprecipitation procedures will now allow us to define these relationships for six well defined secretory proteins: trypsinogens 1 and 2, chymotrypsinogens 1 and 2, procarboxypeptidase 1, and amylase. As a major effort in this project period we will further develop and refine the techniques for ultramicrostructural analysis of radiolabeled pancreatic presecretory, prosecretory and secretory proteins eluted from appropriate one-dimensional or two-dimensional gels. These techniques will include (a) radio amino acid analysis, (b) radiopeptide mapping, and (c) radioprotein sequencing. Using a combination of these techniques we will examine the early events in the biosynthesis of the neutral trypsinogen in the human pancreas in an attempt to explain on a structural basis the very rapid autoactivation kinetics of this zymogen.